In post-natal or adult dermal wound healing, fibroblasts mechanically sense (mechanosense) rigidity and tension that develop in granulation tissue and differentiate into myofibroblasts. Myofibroblasts generate large contractile forces that excessively contract and remodel the ECM resulting in scarring and fibrosis. In contrast, injured skin in the mammalian fetus heals scarlessly without myofibroblast involvement suggesting that smaller cellular forces contribute to regenerative repair. In vivo and in vitro studies have shown that fetal fibroblasts have unique characteristics that may contribute to scarless healing including altered responses to ECM rigidity and defective signaling pathways. However, it remains unclear why fetal fibroblasts retain this distinct phenotype and exhibit differential responses to environmental factors such as ECM rigidity that induce myofibroblast differentiation in adult fibroblasts. Therefore, our overall hypothesis is that fetal fibroblasts d not become myofibroblasts in response to physiologic biomechanical rigidities. We will test this hypothesis with the following Specific Aims: (1) to test the specific hypothesis that fetal fibroblasts have intrinsically altered mechanosensing which leads to ineffective myofibroblast differentiation, (2) to determine how matrix composition influences myofibroblast differentiation since adult and fetal wound healing are characterized by different types of collagen, and (3) to use a sequencing approach to identify molecular differences in fetal fibroblast gene expression that can be used to target myofibroblast differentiation in adult fibroblasts. We are taking an innovative approach by utilizing the mechanical phenotype of fetal fibroblasts as a model for understanding regenerative repair since these cells appear to lack the ability to produce larger cellular forces in response to matrix rigidity which contribute to myofibroblast differentiation an fibrotic healing. We will test our novel concept by using synthetic substrates that mimic the different mechanical stages of wound healing that progressively induce myofibroblast differentiation to isolate the effects of physiologic rigidities and different ECM components. Overall, our goal is to delineate the underlying molecular and physical mechanisms by which fetal fibroblasts differentially mechanosense ECM rigidity by quantifying cellular biomechanical properties relevant to dermal tissue repair. Furthermore, our research plan is designed to uncover potential molecular targets for novel treatment strategies for dermal scarring and fibrosis in post-natal wound healing. These studies are of particular clinical importance since no acceptable anti-fibrotic therapies currently exist and dermal scarring and fibrosis costs billions f dollars of year in medical care and management. In addition, the expected outcomes of our proposed studies are relevant to other fibrosis-related pathologies as well as to the fields of tissue engineering and regenerative medicine.